Loyola University Chicago Loyola eCommons

Master's Theses Theses and Dissertations

1986

Identification of Lactoferrin in the Saliva of Patients with Gingivitis

Hwu-Rang Lin Loyola University Chicago

Follow this and additional works at: https://ecommons.luc.edu/luc_theses

Part of the Dentistry Commons

Recommended Citation Lin, Hwu-Rang, "Identification of Lactoferrin in the Saliva of Patients with Gingivitis" (1986). Master's Theses. 3513. https://ecommons.luc.edu/luc_theses/3513

This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Master's Theses by an authorized administrator of Loyola eCommons. For more information, please contact [email protected].

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1986 Hwu-Rang Lin UBRARY LOYOLA UNIVERSITY MEDICAL CENTER

IDENTIFICATION OF LACTOFERRIN IN THE SALIVA OF PATIENTS WITH GINGIVITIS

by Hwu-Rang lb.in, B.D.S.

A Thesis Submitted to the Faculty of the Graduate School of Loyola University of Chicago in Partial Fulfillment of the Requirements for the Degree of Master of Science

August 1986 DEDICATION

To my parents and my wife, whose loving, devotion, encouragement, and sacrifice made this all possible.

ii ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to the following people: Dr. Patrick Toto, thesis director, for his excellent guidance and encouragement throughout this investigation. To the rest of the members of my co11111ittee, Dr. Anthony Gargiulo and Dr. James Hagen for their assistance and support during this study. Gratitude and special thanks to Mr. Robert Martinez for his excellent technical assistance. To my wife, Marie, for her encouragement, understanding and inspiration during this phase of my education.

iii VITA

The author, Hwu-Rang Lin, B.D.S., is the son of Hwu-Zong Lin and Shou-Ching (Chen) Lin. He was born in Hsin-Chu, Taiwan, R.O.C. on July 22, 1952. His elementary and secondary education was obtained in Hsin-Chu, Taiwan. In September 1970, he entered Taipei Medical College School of Dentistry and received his degree of Bachelor of Dental Science in June 1976. Immediately after he graduated from Taipei Medical College he served two years in the Anny of the Republic of China as a Second Lieutenant, Dental Officer (1976-1978). In May 1978, he was honorably discharged from the Anny. After his release from military service, he worked as a teaching assistant and resident in Taipei Medical College School of Dentistry, Taipei, Taiwan, until 1982. On June 23, 1982 he married Marie Chang in Taipei, Taiwan. In Sep­ tember 1982, he entered Loyola University of Chicago to continue his graduate dental education leading to a degree of Master of Science in Oral Biology. In September 1983, he also entered a two year post-graduate clinical spe­ cialty in Periodontics. On September 24, 1984, Jeffery Gee-Jay Lin the first child of Hwu-Rang and Marie Lin was born. In May 1985, he received a Certificate of Specialty in Periodontics.

iv TABLE OF CONTENTS

Page DEDICATION. . . . ii ACKNOWLEDGEMENTS. iii VITA...... iv LIST OF TABLES...... vi CONTENTS OF APPENDICES. . . . . vii CHAPTER

I. INTRODUCTION...... 1 II. REVIEW OF LITERATURE...... 2 Iron and Infection. . 2 Sources of Lactoferrin...... 5 Properties of Lactoferrin . . . . . 9 The Function of Lactoferrin ...... 10 Alteration of Lactoferrin Levels .. 15

Role of Bacteria in Gingivitis .... 19 Enzyme-linked Immunosorbent Assay 21 III. MATERIAL AND METHOD . 24 IV. RESULTS .... 29 V. DISCUSSION .. 40 VI. SUMMARY AND CONCLUSION. 45 BIBLIOGRAPHY. . • . . . . 47 APPENDIX 55

v LIST OF TABLES

Table Page

1. Gingival Index for the Experimental Group .. 31 2. Gingival Index for the Control Group ..• 32 3. Gingival Index (Mean+ S.D.), comparison between two groups • . . . • . . • . • ...... 33 4. Probing Pocket Depth (rrvn) for the Experimental Group...... 34 5. Probing Pocket Depth (rrvn) for the Control Group. . 35 6. Probing Pocket Depth (Mean+ S.D., mm), comparison between two groups . . . . . • . . . . 36 7. Lactoferrin Concentrations (mg%) for the Experi- mental Group ...... 37 8. Lactoferrin Concentrations (mg%) for the Control Group. • . • • ...... 38 9. Lactoferrin Concentrations (Mean+ S.D., mg%), comparison between two groups. • ...... 39

vi CONTENTS OF APPENDICES

Page Periodontal Charting of Patient #1 . . . . 56 Periodontal Charting of Patient #2 ...... 57 Periodontal Charting of Patient #3 . . . . 58 Periodontal Charting of Patient #4 ...... 59 Periodontal Charting of Patient #5 ...... 60 Periodontal Charting of Patient #6 . . . . . 61 Periodontal Charting of Patient #7 ...... 62 Periodontal Charting of Patient #8 ...... 63 Periodontal Charting of Patient #9 ...... 64 Periodontal Charting of Patient #10 . . . . 65 Periodontal Charting of Subject #11 66 Periodontal Charting of Subject #12 ...... 67 Periodontal Charting of Subject #13 ...... 68 Periodontal Charting of Subject #14 . 69 Periodontal Charting of Subject #15 ...... 70 Periodontal Charting of Subject #16 • . . . . . 71 Periodontal Charting of Subject #17 ...... 72 Periodontal Charting of Subject #18 • ...... 73 Periodontal Charting of Subject #19 • . . . . 74 Periodontal Charting of Subject #20 • ...... 75

vii CHAPTER I

INTRODUCTION

In 1939 a red protein was discovered in bovine milk by Sorensen and Sorensen. This protein has been given various names, such as lactoferrin, lactotransferrin, lactosiderophilin or red milk protein. Because of its absence in serum, lactoferrin has been regarded as a specific milk protein. However, by using inmunological methods it has also been possible to demonstrate the presence of lactoferrin in various external fluids. Elevations in the level of lactoferrin in secretions have been re­ ported in several infectious diseases, such as mastitis, pancreatitis and salivary gland diseases. Therefore, lactoferrin might play an important role in protection of epithelial tissues from infection. The presence of lactoferrin in saliva suggests that it may be important in the control of oral disease processes, such as gingivitis, periodontitis and dental caries. The purpose of this study was to detennine if there is any differences in the concentrations of lactoferrin in saliva between patients with gingi­ vitis and clinically healthy individuals without gingivitis, using the enzyme­ linked inmuno-sorbent assay (ELISA} technique. Changes in the level of lac­ toferrin after the treatment for the patients with gingivitis was also de­ tennined.

1 CHAPTER II

REVIEW OF LITERATURE

Iron and Infection Iron is required by all living cells, including bacteria. According to Weinberg (1978), bacteria need approximately 0.4 to 4.0 uM iron for growth. In the context of bacterial growth in man iron not freely available to micro­ organisms. Iron content of an average man is 4.5 gm (Bell et al., 1953). It is tightly bound to hemoglobin, myoglobin, ferritin and hemosiderin. Although the total iron concentration in manmalian body fluids is 2 x 10-5M (Grif­ fiths et al., 1980), almost all of the iron is associated with iron-binding and transport proteins such as transferrin in plasma and lactoferrin in mu­ cosal secretions. Only 10-18M free-iron is available in body fluids (Bullen et al, 1978; Bullen et al, 1981). This concentration is far lower than the bacteria need for growth. Furthermore, mammals, birds, reptiles and verte­ brates can make metabolic adjustment during infection to withhold the essen­ tial iron from invading bacteria. Such invaded hosts promptly become hypo­ ferrimic by halting intestinal assimilation and by increasing the quantity in storage of iron in the liver (Weinberg, 1974). Prior stationing of host iron-binding proteins at potential sites of bacterial invasion and increased synthesis of host iron-binding proteins are also two possible antibacterial protective mechanisms (Weinberg, 1978). The ability of the host to withhold iron from microbes is called "nutritional

2 3 immunity". The term was first used by. Kochan in 1973. Therefore, it is the ability of the microbes to acquire the iron essential for their growth and metabolism that may determine the nature of the host-parasite interactions. One strategy to obtain iron by the successful pathogens is that they must rapidly adapt to the low-iron environment. The successful pathogens produce powerful iron-chelating agents that can remove iron from iron-binding pro­ teins. Because of extreme insolubility of ferric ion at neutral pH, aerobic and facultative microbes can synthesize either phenolates, which are deriva­ tives of 2,3-dihydroxybenzoic acid, or hydroxamates, which are derived from hydroxamic acid. Phenolates and hydroxamates in bacteria serve to solubilize

11 11 and assimilate the iron. These compounds are known as siderophores • Anaero­ bic bacteria do not need to produce such iron-binding compounds, as they pro­ duce transport ligands that have a selective affinity for ferrous ion (Lank­ ford, 1973}. Many studies on experimental infection have shown that iron enhances bacterial virulence (Weinberg, 1978; Bullen, 1981; Findelstein et al., 1983; Hill, 1978; Bullen et al., 1968} in such strains as Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, Neisseria meningitidis, Neisseria gonor­ rhoeae, Bacteroides melaninogenicus and Staphylococcus aereus. Bornside et al. (1968) have shown that both£. coli and hemoglobin are necessary to produce lethal effect in infections of the peritoneal cavity. Although f. coli is a common inhabitant of the gut, it usually is not regrad­ ed as a highly virulent pathogen. They also found that lysed red cells were as effective as hemoglobin with I· coli in producing a lethal effect. 4

Bullen et al. (1968) further demonstrated that it is the iron in hemo­ globin that is responsible for stimulating bacterial growth. They reported that 1· coli possess a binding site for the iron binding heme molecule from which it gains selective growth advantage, when hemoglobin is available. Based upon a number of clinical studies there is evidence of a correl­ ation between the increase in the hemoglobin concentration in serum and sus­ ceptibility to infection. Bullen (1981) concluded that it is the liberation of heme compounds that can enhance clinical infections. Gingival bleeding commonly accompanies gingivitis and periodontitis. In such diseases the excess hemoglobin that was released could enhance the virulence of the bacteria in the gingival sulcus. Buhler and Muhlemann (1975) recorded the degree of gingivitis and col­ lected the stimulated mixed saliva from 50 subjects, before and after a 10- day period of intensified oral hygiene and measured the iron content in the saliva. They found that the use of methods to improve oral hygiene can re­ duce gingival inflammation and salivary iron content. Gingival inflammation was significantly but not strongly (r= 0.51) correlated with salivary iron content. Mukherjee (1985) measured the concentration of iron in crevicular fluid from sites in gingivitis and periodontitis in human subjects. The results showed that the concentrations of iron in crevicular fluid collected from periodontitis sites were significantly higher than those collected from gingivitis sites (5.196 vs. 3.042 mg/L). These concentrations (5.196 and 3.042 mg/L) were higher than the concentration in serum (about 1.26 mg/L) as 5

reported by Skypeck and Joseph (1981). Mukherjee concluded that this high iron concentration in crevicular fluid might play an important role in the growth and virulence of microorganisms of subgingival plaque and the initia­ tion of active periodontitis. Becraft et al. (1977) performed in vitro studies to investigate the relationship between the administration of intramuscular iron-dextran for prevention of iron deficiency and the susceptibility to I· coli infection. They found that an increased incidence of.£_. coli meningitis occurred after iron-dextran treatment. They reported in these cases the sepsis was due to excess iron concentration in serum. Excess iron causes inhibitory ef­ fects on leukocytes chemotaxis and markedly reduced bacteriostatic effects against.£_. coli. Bullen and Wallis (1977) also have demonstrated that such reversal of the bactericidal effect of polymorphonuclear leukocytes occurs by a fer­ ritin-antibody complex. They found that there is no effect on abolishing the bactericidal power of polymorphonuclear leukocytes by exposing them to iron salts unless high concentrations of iron are used. They further re­ ported that if the iron is presented in the form of a ferritin-antibody complex it is readily phagocytosed by neutrophils, and that the introduction of iron in this form, results in a viable bacterial count 346 times that of the controls at 8 hours.

Sources of Lactoferrin Lactoferrin was first described in bovine milk by Sorensen and Soren­ sen in 1939. Johansson in 1958 found the similar iron-binding protein in 6 human milk. Montreuil et al. in 1960 described a method of isolating and pruifying lactoferrin from human milk. Biserte et al. in 1963 first demonstrated lactoferrin in sputum and could not identify it in saliva. They considered lactoferrin to be bron­ chial origin. Using i11111unohistochemical method by staining sections of biopsy spe­ cimens with fluorescein-labelled antiserum against lactoferrin, Masson et al. (1965, 1966) further demonstrated the presence of lactoferrin in all bronchial glands. Both serous and mucous cells appeared to be involved in the production of lactoferrin. Masson et al. (1965b) reported the same iron-binding protein in human saliva. A bronchial origin for the salivary lactoferrin is not at all im- possible, since bronchial mucus is known to reach the pharynx owing to the propulsory activity of the ciliated respiratory epithelium. However, Masson et al. (1966) have obtained further invnunohistochemical evidence for the occurrence of lactoferrin in the salivary glands. Masson et al (1966, 1966b) demonstrated that lactoferrin is present in a large number of human secretions: (1) respiratory secretions, e.g. nasal and bronchial fluids; (2) tears; (3) secretions from the glandular appendages of the digestive tract, e.g. saliva, bile and pancreatic juice; (4) genital secretions, e.g. seminal fluid and cervical mucus and (5) urine. The inmunohistological localization of lactoferrin in the mucosa of a variety of normal human tissues was investigated using specific fluores- . ceinated antisera by Tourville et al. (1969). They found that the lacto- ferrin was much less ubiquitous in the epithelial cells of the various 7

tissues studied and appeared to be restricted pri~arily to the acinar ep­ ithelium of the bronchial mucosa, parotid and submaxillary salivary glands (serous acinar cells only}; and was also found in renal tubular cells. Reitano et al. (1980} used an i11111unoperoxidase technique to study the distribution of lactoferrin in human salivary glands from autopsy tissues. Such tissues were fixed in Carnoy's fluid for the optimal preservation of lactoferrin antigenicity. They found that specific staining was seen in the intralobular ducts of all salivary glands but never in the interlobular ducts. Also, acinar lactoferrin was detected in most serous demilunes of the mixed glands and in some, but not all, acinar cells of the pure serous glands. However, no lactoferrin was detected in the acinar cells of the pure mucous glands. They also found that polymorphonuclear leukocytes were the only additional cells containing lactoferrin in the oral tissues studied. Moro et al. (1984} used an inmunofluorescence technique to examine the distribution of lactoferrin in human minor salivary glands. Lactoferrin was found in serous acini, demilunes, intercalated and intralobular ducts. They concluded that in addition to major salivary glands and gingival crevicular fluid, the minor salivary glands are also sources of lactoferrin in whole saliva. The reported findings cited above have created the impression that lactoferrin is chiefly an epithelial secretion product. Masson et al. (1968} studied the human uterine cervix and found that lactoferrin originated from a particular cell type adjacent to the epithe- 1 ium, possibly white cells. Also, their investigations on guinea pig tis­ sues have shown that the spleen and bone marrow also are rich sources of lactoferrin. They concluded that glandular epithelia are not the only 8

sources of lactoferrin in mucosal and external secretions, but certain blood cells may also be implicated in the production of this protein. Masson et al. (1969) have demonstrated that lactoferrin is one of the major proteins present in human and guinea pig neutrophilic polymorphonu­ clear leukocytes. This identification was based on a comparison of its electrophoretic, antigenic, and iron-binding properties with the correspond­ ing properties of the same protein isolated from human and guinea pig milk. Immunochemical quantitations showed that lactoferrin occurs in human neutro­ phil ic leukocytes at the concentration of 3 ug per 106 cells. Immunohisto­ chemical data indicated that lactoferrin first appears in myeloid cells at the stage of the promyelocyte. Baggiolini et al. (1970) further demonstrated that the lactoferrin of rabbit heterophil leukocytes has an intracellular distribution almost identi­ cal to that of alkaline phosphatase. The parallelism between lactoferrin and alkaline phosphatase was observed both in the sedimentation rate and in equilibrium density. They concluded that lactoferrin in the heterophil leukocytes belongs to the same granules as does alkaline phosphatase. They are specific (secondary) granules in polymorphonuclear leukocytes. Using direct and indirect immunofluorescent staining, Green et al. (1971) found that lactoferrin was located in the nuclei and/or nuclear mem- brane of mature human polymorphonuclear neutrophilic leukocytes. However, it is absent from lymphocytes, monocytes and eosinophiles. While Masson et al. (1969) have reported that lactoferrin was located in the cytoplasm of human polymorphonuclear leukocytes. Green et al. explained the discrep­ ancy in the localization of lactoferrin in the nucleus and cytoplasm could 9

be due to using different fixative and lactoferrin from different species. Leffell and Spitznagel (1972) also found that lactoferrin is contained in cytoplasmic granules of human polymorphonuclear leukocytes. Upon centri­ fugation, it sediments in a band of granules that also contain 50% of the lysozyme activity. Their findings accorded with those of Masson et al. and Baggiolini et al. (1970).

Properties of Lactoferrin Lactoferrin has a single-chain.structure with the molecular weight 75,000 - 80,000 (Querinjean et al. 1971; Castellino et al., 1970). Each molecule has two specific metal-binding sites. One bicarbonate ion will bind tightly for each metal ion complexed to the protein (Masson and Heremans, 1968b). Teuwissen et al. (1972) by means of a spectrophotometric titration method, found that the binding of Fe3+ by apolactoferrin or apotransferrin involves three tyrosyl residues per metal ion. Gordon et al. (1963) had determined the amino acid composition of lactoferrin in bovine milk, and found that the basic character of the pro- tein is evidenced by an excess per molecule of 27 cationic residues over anionic residues. Some investigators have insisted on the resemblance be­ tween lactoferrin and the iron-binding protein from serum, transferrin, be­ cause both proteins have a single-chain structure with the same molecular weight (Querinjean, 1971). They also have the same metal-chelating proper­ ties, as both are able to bind two atoms of iron. For each Fe3+ taken up, one moleucle of bicarbonate is incorporated (Masson and Heremans, 1968b) and three protons are released (Querinjean, 1971). However, the two proteins 10

appear to differ widely from one another, since no immunologic cross-reac­ tivity has been demonstrated between serum transferrin and lactoferrin of the same species (Montrenil et al., 1960; Blanc and Isliker, 1961). Amino acid compositions have also failed to show similarities (Gordon et al., 1963; Blanc et al., 1963). Furthermore, the iron-binding sites of lacto­ ferrin are more stable at pH values below 4.0 than those of transferrin (Johansson, 1960). Aisen and Leibman (1972) had shown that the binding con­ stant for iron at pH 6.4 - 6.7, was 300 times larger for lactoferrin than for transferrin.

The Function of Lactoferrin It has been reported by Schade (1946) that iron-free serum transferrin displays marked bacteriostatic effect on microorganisms. In order to assess whether lactoferrin has the similar properties, Masson et al. (1966b) per­ formed a number of experiments in which solutions of lactoferrin of various degrees of saturation with iron were tested for their inhibiting capacity. They found a clear inhibitory effect by unsaturated lactoferrin on the Staph­ ylococcus albus, Staphylococcus aureus and Pseudomonas aeruginosa. This in­ hibitory activity had disappeared when ferrous ammonium sulfate had been added to the lactoferrin. They suggested that iron-unsaturated lactoferrin is bacteriostatic in action. Reiten and Oram (1967) found that lactoferrin is very heat stable (90°C for 60 minutes) but is inactivated by trypsin. They also reported that purified lactoferrin inhibited the growth of Bacillus stearothermophilus and Bacillus subtilis and that its inhibitory activity is suppressed by 11

ferrous ions. Oram and Reiter (1968) further extended their study and again have shown that the bacteriostatic action of lactoferrin was suppressed by Fe2+ 2+ 2+ .2+ 2+ and enhanced by Zn ·' Co , N1 , and Cu . Similar findings were reported by Kirkpatrick et al. (1971), by using radial i11111unodiffusion method to measure the content of lactoferrin in par­ otid flui.ds and leukocytes from patients with chronic mucocutaneous candi­ diasis and normal subjects. They found that there were no differences in the concentrations of lactoferrin in the salivas from two groups. The iron­ unsaturated protein markedly impaired replication of the yeast, and the growth-inhibitory property was lost when lactoferrin was saturated with iron. Bullen et al. (1972) found that human milk with unsaturated iron-bind- ing lactoferrin had a powerful bacteriostatic effect on Escherichia coli. The bacteriostatic properties of milk were abolished if the iron-binding proteins were saturated with iron. They also studied guinea pig milk, and found that newly born guinea pigs fed on an artificial diet and dosed with E. coli had higher counts of E. coli in the intestine than normally suckled animals. They suggested that iron-binding proteins in milk may play an important role in resistance to infantile enteritis caused by E. coli. Reiter et al. {1975) found two strains of E. coli that were not in­ hibited by undiluted colostral whey or milk. However, the colostral whey became bacteriostatic for E. coli after dialysis or dilution in Kolmer saline and the addition of precolostral calf serum or lactoferrin. The lack of inhibition of E. coli in undiluted whey is due to the high 12

concentration of citrate in colostral whey and milk. It is suggested that citrate competes with the iron-binding proteins for iron and makes the citrate-iron complex available to the bacteria for growth. Bishop et al. (1976) reported in their in vitro study that as little as 0.02 mg of apo-lactoferrin per ml deionized distilled water in marked inhibition of all mastitis-causing coliform bacteria. Growth inhibition was lost if iron-saturated lactoferrin or iron plus apo-lactoferrin was added to the synthetic medium. They also found that apo-lactoferrin at 20 mg/ml was bactericidal for E.coli. The addition of apo-lactoferrin plus citrate re­ sulted in loss of growth inhibition, however, the molar ratio (citrate to apo-lactoferrin) was found to be more important than the absolute concentra­ tion of either component. Because a ratio of 75 resulted in 50% growth in­ hibition of E.coli, whereas ratios of 300 and greater resulted in less than 10% growth inhibition. The antimicrobial action of lactoferrin has been attributed to the ability of depriving the essential iron. And the reversibility of this an­ timicrobial effect, when an excess of iron is supplied to the nutritionally deprived organisms, suggests a simple bacteriostatic effect for lactoferrin. But Arnold et al. (1977) showed Streptococcus mutans and Vibrio cholerae, but not Escherichia coli, were killed by incubation with purified human lac­ toferrin. The resistance of E. coli to the bactericidal action of lacto­ ferrin in their study, could be due to the production of iron chelators (enterochelins). The ability to synthesize iron chelators has been sug­ gested as a significant virulence factor for several enteropathogenic strains of E. coli. The investigation by Bullen et al. (1972) and Bishop et al. 13

(1976) have suggested that synthesis of enterochelins allows the bacteria to utilize lactoferrin. Bullen and Annstrong (1979) reported rabbit polymorphonuclear leuko­ cytes contain the iron-binding protein lactoferrin and can rapidly phago­ cytose and destroy Pseudomonas aeruginosa. If the cells are exposed to a ferritin-antibody complex, large amounts of this are phagocytosed by poly­ morphonuclear leukocytes and appear in the cytoplasmic granules and phago­ somes. This leads to saturation of the cellular iron-binding protein with Fe, the bactericidal power of the cells is greatly reduced with the result that some phagocytosed bacteria survive and eventually grow and destroy the cells. An apoferritin-antibody complex used as a control is also phag­ ocytosed by polymorphonuclear leukodytes but has no effect on the bacteri­ cidal power of the cell. Arnold et al.(1980) extended their study on bactericidal activity of human lactoferrin. The sensitivity of a variety of miroorganisms to human lactoferrin was investigated. Some of the microorganisms were clinical isolates from 4-day supragingival plaque samples. They found that suscep­ tible microorganisms includes Gram-positive and Gram-negative microbes, rods and cocci, facultative anaerobes, and aerotolerant anaerobes. Also, they found organisms of the same species and even of the same strain can differ in susceptibility to lactoferrin. They suggested that accessibility of the microorganisms to the lactoferrin target site may account for differences in susceptibility. Arnold et al. (1981) studied the influence of the metabolic state of the bacteria, the incubation temperature, pH, and the presence of metal 14 ions on the kinetics of killing Streptococcus mutans by lactoferrin. They found that after exposure to lactoferrin, a 15-minutes lag period occurred before the initiation of killing, indicating that a two step process i~ in­ volved in lactoferrin killing. Cultures harvested during the early exponen­ tial phase were very sensitive to lactoferrin, whereas cultures harvested in the early stationary phase were markedly more resistant. Also, the rate of killing was dependent on temperature. At 37°C, there were no detectable colony-forming units (CFU) after 1 hour of incubation. However, when the incubations were carried out at 23°C or at 4°C, the rate of killing was markedly reduced. Additionally, bactericidal activity was enhanced under the slightly acid environment. This indicated that the reduction of the killing activity at neutral pH and the loss of killing under slightly alka­ line condition might be due to the significant conformational changes of lactoferrin that occur with increasing pH. Furthermore, exogenous ferrous or ferric ions did not reverse or prevent lactoferrin killing. Arnold et al. (1982) compared the bactericidal effects of lactoferrin on Streptococcus mutans with the bacteriostatic effects on iron deprivation. They found that growth and macromolecular synthesis (DNA, RNA and protein) were inhibited by incubation of S. mutans with purified human lactoferrin. Their early studies (1980, 1981) have shown that lactoferrin binds to the surface of susceptible bacteria, and this binding could result in the in­ hibition of growth by blocking some essential transport sites, resulting in nutritional deprivation of the bacteria. If this is true, then removal of the surface-bound lactoferrin should reverse this inhibition. But, in the experiments (1982}, even after using a variety of washing agents to remove 15 the bound lactoferrin from the cell surface, the viability could not be re­ stored. They concluded that this inhibition of metabolism and rapid loss in viability observed with lactoferrin treatment suggest that lactoferrin has a direct bactericidal effect on S. mutans that can not be attributed to simple iron deprivation. An alternative possible suggestion was that the binding of lactoferrin to the bacterial cell surface triggers a second pro­ cess that results in the irreversible cell death.

Oseas et al. (1981) found that PMNs released the specifi~ granule pro­ duct lactoferrin more rapidly in response to chemotatic stimuli, which cor­ related with promotion of PMNs aggregation and enhanced PMNs adherence. They suggested that PMN lactoferrin serves an autoregulatory role to retain PMNs at inflanmatory sites to amplify the inflanmatory response. Ambruso and Johnston (1981) reported that iron-saturated lactoferrin from human colostrum and human neutrophils enhances the generation of the bactericidal hydroxyl radical. This product may result from the competetive ability of lactoferrin to obtain iron from bacteria. They suggested that in addition to antimicrobial function, lactoferrin could play an important role as regulator of hydroxyl radical production during phagocytosis.

Alteration of Lactoferrin Levels Estevenon et al. (1975) used an inmunodiffusion technique to test the presence of lactoferrin in the duodenal juice of patients with chronic cal­ cifying pancreatitis (CCP). They found that almost all patients with CCP and 7% of control subjects without CCP have shown lactoferrin in duodenal juice. They suggested that the lactoferrin test could have a certain value 16

in the diagnosis of CCP, especially in the differential diagnosis between CCP and other pancreatic diseases. Colomb et al. (1976) further extended their study in 1975. By using the i1T1Tiunodiffusion techniques, lactoferrin was present in the pathological pancreatic juice only. However, when indirect ilTITlunofluorescence method was used, the fluorescence was found in all samples, including normal pancreatic tissue. The difference between these two techniques were explained by the different sensitivity and these results indicated that lactoferrin is a pro­ tein of pancreatic secretions. Fedail et al. (1978) used radioimmunoassay to measure the concentra­ tion of lactoferrin in pure pancreatic juice from the patients with chronic pancreatitis and control subjects. They found that mean concentrations of lactoferrin were 2310 mg/ml (range 870 - 11,4000) in the patients with chron­ ic pancreatitis and 128 mg/ml (range 30 - 390) in control subjects. They suggested that the measurement of lactoferrin concentration in pure pan­ creatic juice may be useful in the diagnosis of pancreatic diseases. Lacto­ ferrin seems is a sensitive marker of chronic pancreatitis because lacto­ ferrin concentrations in pancreatic juice are specifically elevated in these patients. Harmon et al. (1975) determined the lactoferrin concentration in milk by electroimmunodiffusion assay from normal lactating cows and cows with mastitis. They found that the mean concentration of lactoferrin in normal group was 0.35 mg/ml. In contrast, that found in the mastitis group from 0.55 mg/ml on day 1 to 1.89 mg/ml by day 3. Harmon et al. (1976) also found that experimentally induced Escherichia coli infection of bovine marrmary 17

gland resulted in a 30-fold increase in lactoferrin concentration in mammary secretion by_90 hours post-inoculation, and 4-fold increase in total daily production of lactoferrin by 264 hours post-inoculation. They suggested that the increased lactoferrin production may be a result of a specific re­ sponse of secretory tissue to inflammatory agents. Tabak et al. (1978, 1978b) examined the levels of lactoferrin in the parotid secretions of groups of patients (a) with chronic recurrent paro­ titis, (b) with complaints of dry mouth but no evidence of disease, (c) with suspected or confirmed Sjogren's disease, (d) with sarcoidosis and (e) with diabetes. They found that lactoferrin values in parotid saliva during the active phase of parotitis were elevated from two to twenty times normal. The values for the recovery phase of parotitis were lower than in the active phase, but still two to ten times normal. Elevations in lactoferrin were also noted in five of six patients with Sjogren's disease, but not in sub­ jects with sarcoidosis, diabetes or "dry mouth" without sialographic changes. In 1971, Kakizaki et al. reported a direct relationship between ex­ perimentally induced pancreatic disorders and alterations of histology and function of the parotid glands. Later, they developed a new test on parotid saliva and emphasized the usefulness of the saliva test for the diagnosis of pancreatic diseases (Kakizaki et al., 1976). In order to find out if lactoferrin levels in parotid saliva are ele­ vated in chronic pancreatitis, Durr et al. (1982) and Benini et al.· (1983) measured the concentration of lactoferrin in parotid saliva from patients with pancreatitis and controls. They found that there was no difference in the lactoferrin between these two groups. They concluded that the elevation 18 of lactoferrin concentration is confined to the exocrine pancreas and do not affect salivary glands in chronic pancreatitis. Fridman et al. (1983) using rocket immunoelectrophoresis to study the • concentrations of lactoferrin and lysozyme in the crevicular fluid from patients with gingivitis, adult periodontitis, localized juvenile periodon­ titis and normals. They found that lactoferrin in the normal group was sig­ nificantly lower than those in gingivitis group (0.63 vs. 1.5 ug/ul) but did not show significant differences between gingivitis, adult periodontitis and localized juvenile periodontitis. Konttinen et al. (1984) studied the concentration of salivary lacto- ferrin in patients with Sjogren's syndrome by radioinvnunoassay. The concen­ tration of lactoferrin in unstimulated mixed saliva in Sjogren's syndrome group was 16.28 ± 3.05 ug/mg of protein (range 7.42 - 33.64) and that in sex and age matched normal group was 2.82 ± 0.82 ug/mg of protein (range 0.59 - 7.35). They demonstrated that by using the peroxidase-antiperoxidase stain­ ing method for labial salivary gland biopsy, a strong staining of lactofer­ rin was found in intralobular and interlobular ducts. Their results indi- cated that the concentration of unstimulated, mixed salivary lactoferrin is a useful diagnostic tool for the diagnosis of Sjogren's syndrome. They sug­ gested that abnormal salivary duct secretion and/or reabsorption is the reason for high salivary lactoferrin values. Muratsu and Morioka (1985), in order to study the mechanisms of in­ creased susceptibility to oral infection in diabetics, examined the level of salivary antibacterial factors; including lysozyme, lactoperoxidase and and lactoferrin in diabetic hamsters. They found that although lysozyme 19 activity decreased by 56% and lactoperoxidase activity decreased by 53% in diabetic hamsters, there was no significant difference between the strepto­ zotocin induced diabetic and non-diabetic hamsters in the amount of salivary lactoferrin. Also, before insulin treatment the ratio of lactoferrin to total protein increased to double the amount of that of the control hamsters. After the insulin treatment, the ratio of lactoferrin to total salivary pro­ tein reverted to nonnal values.

The Role of Bacteria in Gingivitis Withdrawal of tooth-brushing in healthy human experimental subjects re­ sults in the accumulation of microbial plaque on the teeth and the develop­ ment of gingivitis (Loe et al., 1965; Theilade et al., 1966). If the bac­ terial plaque is removed by scaling, root planing and gingival curettage, the gingivitis is reversible. It indicates that bacteria are casually re­ lated in gingivitis (Krygier et al., 1973). Listgarten (1976) in a study with the electron microscope found that in healthy gingival sulcus a thin microbial layer approximately 60 um thick adhered to the enamel surface of teeth. The cells were predominantly coc­ coid in shape with a majority exhibiting cell wall features compatible with those of Gram-positive microorganisms. According to Socransky (1977), healthy periodontal tissues appear to be associated with a scanty microbial flora that is located almost entirely supragingivally on the tooth surface. Microbial cell accumulations are usually 1 to 20 cells in thickness and are composed mainly of Gram-positive caecal fonns. 20

The microorganisms commonly encountered in such sites include Strep­ tococcus mitfs, Streptococcus sanguis, Staphylococcus epidennidis, Rothia dentocariosa, Actinomyces viscosus, Actimyces naeslundii and occasionally species of Neisseria and Veillonella (Listgarten, 1976; Listgarten et al., 1975; Socransky et al., 1977; Slots, 1977). According to Slots (1979), a scant microbial flora dominated by Gram­ positive microorganisms (85%), usually Streptococcus and facultative Actin­ omyces species is found in the healthy gingival sulcus. In the gingivitis sites on teeth, Listgarten (1976) found that bac­ terial deposits appeared to be thicker than that found in the nonnal samples, about 0.4 mm in thickness. The bacterial samples comprised a wide variety of microorganisms, including coccoid as well as filamentous fonns, and those with Gram-positive and variety of Gram-negative cell wall patterns. Also, flagellated bacteria and spirochetes were not uncommon in gingivitis samples. According to Socransky (1977), in experimental gingivitis there is an increase in the total mass of plaque and cell layers which often extend to 100 to 300 cells in thickness. There is an increase in proportions of mem­ bers of the genus Actinomyces. This group of microorganisms tends to be the dominant genus associated with supragingival plaque, frequently comprising 50% or more of the isolates (Listgarten et al., 1975; Loesche and Syed, 1975; Syed et al., 1975). In long standing gingivitis, approximately 25% of the microbial flora may be Gram-negative, including species of Veillonella, Vibro (CampYlogacter) and Fusobacterium. The Gram-negative cells appear to be located primarily on the surface of the bacterial plaque in subgingival sites (Listgarten, 1976; van Palenstein Heldennan, 1975). 21

According to Slots {1979), the development of gingivitis is accom­ panied by a marked increased in the total number of Gram-negative micro­ organisms. Fusobacterium nucleatum, Bacteriodes melaninogenicus ~· inter­ medius, Haemophilus species, and other Gram-negative microorganisms com­ prised about 45% of the total gingivitis isolates. Streptococcus and facul­ tative and anaerobic Actinomyces species constituted the majority of the Gram-positive gingivitis isolates. According to Page and Schroeder {1981), gingivitis and periodontitis in man and other animals, without exception, is caused by bacteria. How­ ever, the mere presence of bacteria, even the presence of specific patho­ gens is insufficient. They emphasized that an interaction of bacteria with the host's response systems is essential of the disease is to become estab­ lished and progress. This indicates that a healthy gingiva is the result of an equilibrium between pathogenic factors from the gingival crevice flora and the natural defense of the adjacent tissues. If the nature of gingival crevice flora alters either by an increase in the number of organisms or by a shift to a more virulent bacterial population, the equilibrium might be broken and inflanmation and destruction of tissues will follow.

Enzyme-linked Inmunosorbent Assay Antibodies and antigens labeled with fluorescent dyes or isotopes have been used extensively for inununodiagnosis over the past two decades. However, they do have some disadvantages. Inmunofluorescence, usually de­ pends on subjective assessment of the end result, is time-consuming and is not easily automated so it can be used for only small batches of tests. 22

Radioill1Tlunoassay is particularly suitable for large-scale operations but the short shelf-life of the reagents, the expensive equipment, and because it carries some risks, has tended to exclude radioill1Tlunoassay from many small laboratories. These considerations have led workers to search for alternative labels for antiboides or antigens {Boller et al., 1976; Voller et al., 1978). Enzyme labeled reactants are safe, have long shelf life and yield objective results with the same sensitivity as radioirnmunoassay, yet can be used with relatively cheap, simple equipment. According to Engvall et al. {1971), by using enzyme-linked irnmunosorbent assay {ELISA) 1 - 100 ng/ml of the antigen could be determined. The basic ELISA test depends on two assumptions: (1) that antigen or antibody can be attached to solid-phase support yet retain ill1Tlunological activity and (2) that either antigen or antibody can be linked to an enzyme and the complex to retain both immunological and enzymatic activity. Accord­ ing to Voller et al. {1976) the double antibody 11 sandwich 11 for detection of antigen and the indirect method for detection of antibody are the most use­ ful in practice. It is clear from the work of Cantarero et al. {1980), Fields, et al. {1983) and Kemeny et al. (1985) that inmobilization of proteins to plastic material is dependent on the nature of the protein and the conditions used for coating. Kemeny et al. (1985) found that prolonged incubation of the antibody with the plates resulted in an increased capacity for binding antigen. Also, the rate of antigen bound to the antibody-coated plates was 23 much faster than that for the antibody alone bound to the plates, and the amount of antigen bound to the antibody-coated plates did not increase after 1 hour. A serious problem limiting the measurement of specific antigen is the high non-specific antibody binding to the plates. Urbanek et al. (1985) found that the rate of antigen bound to antibody-coated plates showed down after 3 hours incubation while the non-specific binding increased progres­ sively with longer incubation. Also, the rate of this binding was higher at 37°C than 21°C or 4°C, but this was largely due to increased non-specific binding at this temperature. They suggested that the best separation of specific antibody binding from non-specific binding was to incubate the plates at 4°C. Thus, in this study we reduced the incubation time to 1 hour and re­ duced incubation temperatures to 4°C. Also, we used 0.1% bovine serum al­ bumin (BSA) and 1% nonnal rabbit serum (NRS) as blocking agent. Also, Tween- 20 was included in all washing steps to prevent unspecific binding. CHAPTER III

MATERIAL AND METHOD

Selection of Patients Ten adult clinical patients with gingivitis, three females and seven males, ranging in age from 20 to 31 were selected from the Department of Periodontics, School of Dentistry, Loyola University of Chicago, based on the following clinical criteria described by Listgarten in 1976: (1) Gin~i­ val Index (GI): 2 - 3 ; (2) Pocket depth less than 5 mm; (3) Absence of radiographic evidence of bone loss. In addition, a group of age and sex­ matched clinically normal control subjects with healthy gingiva (3 females and 7 males, ranging in age from 23 to 31) were selected from dental stu­ dents at the School of Dentistry, Loyola University of Chicago. None of the participants had received a prophylaxis within the last six months. They were not taking any medication and did not show any evi­ dence of systemic diseases or salivary gland diseases. The female partici­ pants were neither pregnant nor taking oral contraceptives. Pocket depth was measured to the closest millimeter by means of a standard periodontal probe. Bone loss was considered to be absent, meaning that the alveolar crest is within 2 mm of cementoenamel junction(CEJ). The severity of gingival inflammation was assessed by the criteria of the Gingi­ val Index of Loe and Silness (1963) as modified by Loe (1967). Four gingi­ val areas, buccal, lingual gingival margin and interdental papillae, of all

24 25 teeth were given a score from zero to three as follows: 0: normal (absence of inflammation) 1: mild inflammation (slight change in color, little change in texture, no bleeding on probing.) 2. moderate inflammation (moderate glazing, redness, edema and/or hypertrophy, bleeding on probing.) 3. severe inflammation (marked redness and edema, hypertrophy, spontaneous bleeding or ulceration.) The scores from the four areas of the tooth were added and divided by four to give the GI for the tooth. Finally, by adding the indices for the teeth and dividing by the total number of teeth examined, the GI for the individual was obtained.

Experimental Design This study was reviewed by the chairman of the Loyola University Medi­ ical Center Institutional Review Board for the Protection of Human Subjects (IRB) before commencement. At the baseline, after signing the consent form, participants were given a clinical examination, and provided saliva samples as subsequently described. Patients with gingivitis received the following treatment: oral hy­ giene instruction, scaling, root planing and gingival curettage. It took four appointments, with a one week interval, to complete the treatment pro­ cedures. Two weeks after the last appointment, the patients came back to clinics for re-examination. At this time, the baseline procedures were re­ peated. for the control group._five weeks after the initial appointment 26 the baseline procedures were repeated, and the study terminated. - Each subject brushed with water to remove plaque and residual debris before saliva collection. All participants spit saliva actively into a funnel connected to a graduated centrifuge tube until 3 ml were obtained. The collected saliva were clarified immediately by centrifugation at 3,675 rpm for ten minutes. 0.1% (about 3 mg) cetylpyridinium chloride (CPC) was added to supernate, and the aggregated material in the supernate was removed. The supernate samples were centrifuged at 3,675 rpm for ten minutes again. The salivary supernatant then were frozen and stored for further tests, as described by Olson et al. in 1985.

Experimental Design

At the baseline (Clinical examination and saliva collection) \ Control/ Group Experimental Group appoiptment 1 .lt {l week) appointment 2 J (1 week) (5 weeks) appointment 3 l (1 week) appointment 4 (2 weeks) ) At the reexamination ~~~~~~~~- (Clinical examination and saliva collection) 27

ELISA Procedures Quantitation of lactoferrin was perfonned by enzyme-linked inmunosor­ bent assay (ELISA) as described below (Dipaola and Mandel, 1980; Kemeny et al., 1985; Urbanek et al.,' 1985): 1. Tissue culture plates (American Scientific Products 24 - 2 ml wells) were employed as solid phase. 2. The wells were coated with specific antibody to lactoferrin by passive adsorption and run in triplicate. 2 ml of rabbit anti­ human lactoferrin (Pel-Freez Biological) at concentration of

10 ug/ml in 0.05 M Na 2co3 buffer (pH 9.6) were added to each well. 3. The coated wells were incubated at 4°C for overnight. 4. The wells were washed four times to remove unbound antibody with tris-buffered saline (TBS). 5. The coated wells were then treated with 0.1% bovine serum albumin and 1% normal rabbit serum in 0.05 MTBS at room temperature for 30 minutes. 6. The wells were washed four times with TBS. 7. 2 ml of lactoferrin, prepared from human standard serum (Calbiochem) at concentrations of 10.3 to 26.9 ng/ml in 0.05 M

TBS were added to triplicate wells to establish a stand~rd curve. 8. 2 ml of test saliva diluted 1:500 with 0.05 MTBS, pH 7.4 were added to the remaining wells. 9. The plates were incubated at 4°C for 1 hour. 10. The wells were washed four times with TBS. 28

11. 2 ml of an enzyme-labeled preparation of the antibody, rabbit anti-human lactoferrin-alkaline phosphatase {Pel-Freez Biologi­ cal), diluted 1/200 with 0.05 MTBS and 0.2% BSA were added and incubated at 4°C for 1 hour. 12. The wells were washed four times with TBS. 13. 2 ml of p-nitrophenyl phosphate, substrate, were added to each well at the concentration of 1 mg/ml in 0.1 M Ethanolamine

{2-Aminoethanol) pH 9.6 containing 10 mM MgC1 2. 14. The wells were incubated at room temperature for 30 minutes. 15. 0.2 ml of 1 N NaOH were added to stop the enzyme reaction. 16. 150 ul solution from each well were transferred to Dynatech plates containing 96 wells. 17. The optical density {OD) of each well was read with Minireader II {Dynatech) at 410 nm. 18. The concentration of lactoferrin was determined by comparison with a standard curve.

The ELISA procedures were performed twice, and the average concentra­ tion of each sample was obtained. The values were analysed by the two sam­ ple student t test in order to establish a statistically significant dif­ ference between the two groups. CHAPTER IV

RESULTS

Clinical Observations

Gingival Index (GI): At the baseline, the gingival index for the experimental group was 1.8 ± 0.2. After the periodontal treatment, the GI was reduced to 1.2 ± 0.3 (Table 1). This was significantly different at the level of 0.001 (Table 3). For the control group, at the baseline and at the reexamination, the GI were

0.2 ± 0.1 and 0.2 ± 0.2 respectively (Table 2), and did not differ signifi­ cantly {Table 3).

Probing Pocket Depth:

At the baseline, the mean probing pocket depth was 2.5 ± 0.2 lll11 for the experimental group, and was 2.0 ± 0.2 mm for the control group {Table 4 and 5). This was significantly different at the level of 0.001 {Table 6). At the reexamination, the mean probing pocket depth was 2.4 ± 0.2 mm for the experimental group, and 2.0 ± 0.2 mm for the control group {Table 4 and 5). This was significantly different at the level of 0.001 (Table 6). However, there was no significant difference between at the baseline and at the reex­ amination either in the experimental or control group (Table 6).

Lactoferrin Concentrations The value for lactoferrin from unstimulated whole saliva from ten

29 30 patients with gingivitis and ten clinically normal individuals, as quanti­ tated by the double antibody sandwich ELISA technique, are shown in Table 7 and Table 8. For the experimental group, at the baseline the mean value was 0.57 ± 0.08 mg%, after the periodontal treatment the mean value was

0.58 ± 0.09 mg%. There was no significant difference. For the control group, at the baseline the mean value was 0.56 ± 0.21 mg%, at the reexamin­ ation the mean value was 0.55 ± 0.15 mg%. There was no significant differ­ ence. Also, no significant differences were found between the experimental and control groups either at the baseline or at the reexamination (Table 9). 31

Table 1

Gingival Index tor the Experimental Group

Patients At the At the baseline reexamination

1 1.6 o.8 2 2.i 1.4 3 1.8 1.4 4 i.7 o.8 5 1.8 o.8 6 2.0 1.5 7 1.9 1.3 8 1.9 1.4 9 1.7 1.3 10 1.6 1.2

Mean 1.8 1.2 S.D. 0.2 0.3 32

Table 2

Gingival Index tor the Control Group

At the At the Subjects baseline reexamination

11 O.J O.J 12 0.2 0.2 1.3 O.J 0 .3 14 0.2 0.2 15 o.o o.o 16 0.5 0.5 17 O.J o.4 18 0.2 0.1 19 o.o o.o 20 0.2 0.2

Mean 0.2 0.2 S.D. 0.1 0.2 33

Table J

Gingival Index ( Mean .:t. S .D.), comparison be~ween two groups

At the At the t p baseline reexamination

Experimental Gr. 1.8 ±. 0.2 1.2 ± O.J 5.83 ~0.001 Control Gr. 0.2 ±. 0.1 0.2 i 0.2 o.oo N.S.* * N.S. = not significantly different. 34

Table 4

Probing Pocket Depth {mm) for the Experimental Group

At the At the Patients baseline reexamination

1 2.4 2.2 2 2.8 2.7 3 2.6 2.6 4 2.5 2.3 5 2.6 2.2 6 2.5 2.4 7 2.6 2.5 8 2.3 2.3 9 2.7 2.6 10 2.4 2.3

Mean 2.5 2.4 S.D. 0.2 0.2 35

Table S

Probing Pocket Depth (mm) tor the Control Group

At the At the Subjects baseline reexamination

11 2.0 2.0 12 1.9 1.9 13 2.2 2.2 14 2.1 2.1 15 2.0 2.0 16 2.2 2.2 17 2.1 2.1 18 2.0 2.0 19 1.7 1.7 20 2.2 2.2

Mean 2.0 2.0 S.D. 0.2 0.2 36

Table 6

Probing Pocket Depth (mean~ S.D., mm), comparison between two groups

At the At the t p baseline reexamination

Experimental. Gr. 2.5 ,!_ 0.2 2.4 :t 0.2 1.4J N.S.* Control Gr. 2.0 :!:. 0.2 2.0 :t. 0.2 O.OO N.S. t 7.14 5.26

p ~0.001 ...( 0 .001

* N.S. = not signi:t'icantly di:t':f'erent. 37

Table 7

Lactoferrin Concent~ations (mg%) tor the Experimental Group

At the At the Patients baseline reexamination

1 0.51 0.46 2 0.53 o.49 J o.68 0.58 4 0.69 0.54 5 0.51 0.63 6 0.53 0.77 7 0.52 0.56 8 o.68 0.58 9 o.46 0.57 10 0.59 o.64 Mean 0.57 0.58 S.D. 0.08 0.09 38

Table 8

Lactoferrin Concentrations (mg%) for the Control Group

At the At the Subjects baseline reexamination

11 0.48 0.51 12 o.49 o.48 lJ o.42 O.J8 14 o.46 o.47 15 o.66 0.61 16 0.50 0.58 17 o.46 0.52 18 o.44 o.48 19 1.13 0.94 20 0.52 o.48 Mean 0.56 0.55 s.n. 0.21 0.15 39

Table 9

Lactoterrin Concentrations (mean ±. S.D., ~), comparison between two groups

At the At the t p baseline reexamination

Experimental Gr. 0.57 s. o.oa 0. 58 :t- 0. 09 0.28 N.S.* Control Gr. 0.56 :t 0.21 0.55 ± 0.15 0.125 N.S. t 0.14 o.6 p N.S. N.S.

* N.S. = not significantly different. CHAPTER V

DISCUSSION

In the present study gingival tissues were assessed for the degree of inflammation by the criteria of the Gingival Index System (Loe and Silness, 1963; Loe, 1967). ·The criteria include gingival tissues pale pink in color, stippled in texture, firm in consistency, and in the absence of bleeding upon probing is considered clinically normal. This criteria received a Gin­ gival Index score of zero. Gingival tissues displaying mild inflammation, slight change in color, slight edema, no bleeding on probing, and received a Gingival Index score one. Gingival tissues displaying moderate inflamma­ tion, redness, edema and glazing, in the presence of bleeding upon probing, and received a Gingival Index score of two. Gingival tissues which displayed severe inflammation, ulceration, and tendency to spontaneous bleeding re­ ceived a Gingival Index score of three. All participants in the control group were considered to be clinically normal, while in the experimental group, the scores were changed from 1.8 at the baseline to 1.2 at the reexamination. This significant change was ap­ parently the result of the scaling, root planing, curettage and oral hygiene instruction as performed in the protocol. The experimental results reported in this study do show the presence of lactoferrin in the whole saliva of the patients with gingivitis and in clinically normal individuals as measured by the ELISA technique. 40 41

Double antibody sandwich ELISA technique indeed can measure the con­ tration of lactoferrin in the whole saliva. However, the procedures used in this study were modified by precipitation of salivary acid mucopolysac­ charides with cetylpyridinium chloride (CPC) in the saliva processing. CPC is a good fixative for acid mucopolysaccharides (Williams and Jackson,

1956). Since 1 - 100 ng/ml of the antigen can be determined by ELISA (~ng­ vall et al., 1971), ELISA is a very sensitive and useful method to measure such small amount of lactoferrin in saliva. The levels of lactoferrin in the saliva of the patients with gingivi­ tis and individuals with clinically normal gingiva do not have significant differences as determined by ELISA method. This suggests that gingivitis does not change the host's response with respect to the lactoferrin level in saliva. After the patients with gingivitis in this study received scal­ ing, root planing, gingival curettage and oral hugiene instruction, the level of lactoferrin in their saliva did not change. This suggests that lactoferrin level in saliva does not respond to the treatment of gingivitis, even when there is a significant clinical improvement in the gingival in­ fla11111ation. Also, in this study, the probing pocket depth in gingivitis group was

2.5 ± 0.2 11111, and 2.0 ± 0.2 11111 in control group (Table 4 and Table 5), it does not suggest a similar effect in chronic periodontitis. It is not known whether there is any change of the level of lactoferrin in the saliva in the patients with chronic periodontitis. In such cases it is observed that they show deeper probing pocket depth and more soft tissue and alveolar bone destruction. A study of salivary lactoferrin levels in patients with 42 chronic periodontitis in order to assess any relevance and in comparison to the findings-reported in this study should be done in the near future. A previous study by Masson et al. (1966) reported the lactoferrin values in stimulated parotid saliva as low as 0.2 to 0.3 mg% by the electro­ phoresis method. Brandtzaeg (1971) reported lactoferrin concentration in stimulated parotid saliva to be about 1 mg% and also noted that unstimu­ lated salivary samples exhibited higher values than stimulated samples. Tabak et al. (1978) used the rocket electrophoresis method and re­ ported a range of 0.5 to 2 mg% lactoferrin in parotid saliva in their con­ trol subjects. Dipaola and Mandel (1980) who also used the ELISA method reported the lactoferrin value 0.81 ± 0.36 mg% in the unstimulated parotid saliva and

0.54 ± 0.26 mg% in the stimulated parotid saliva, in normal human subjects. In their study, the saliva samples were diluted 1:25. In our study the sali­ va samples were diluted 1:500, and indicates that our ELISA techniques is more sensitive than that reported by Dipaola and Mandel. This may be due to the use of different buffer solution, incubation time and incubation temper­ ature. The sources of lactoferrin in human mouth has not been determined. However, it could arise in part from disrupting polymorphonuclear leukocytes and, in part, from epithelial cells that synthesize lactoferrin in the major and minor salivary glands (Masson, 1968; Tabak et al., 1978). The majority of salivary leukocytes continuously migrate into the mouth through the gingival crevice (Wright, 1964; Schiott and Loe, 1970), and 97 - 99% of them are neutrophils (Attstrom, 1970; Raeste, 1972). The 43 lactoferrin concentrations found in the saliva of the patients with gingi­ vitis and clinically normal individuals appear to be the same in this study. This was unexpected as a three-fold increase in the number of leukocytes en­ tering the gingival crevice had been observed during the course of gingivi­ tis (Schiott and Loe, 1970; Attstrom and Egelberg, 1971). This increased supply of lactoferrin could be balanced by the increased flow rate of gingi­ val crevicular fluid in gingivitis (Attstrom and Egelberg, 1971; Alfano, 1974). The saliva contains other antibacterial proteins. Lactoferrin and several other proteins in saliva have been identified as antibacterial a­ gents, including secretory Ig A, the lactoperoxidase-thiocyanate-hydrogen peroxide system, and lysozyme. The concentration of each of these antibac­ terial agents alone may not be sufficient to influence the oral flora. They may need to act synergistically to accomplish that task. Arnold et al. (1979) found that although there was no significant difference in the lacto­ ferrin values in saliva between the patients with inmune dysfunction and con­ trols, the salivary concentrations of lysozyme and lactoperoxidase tend to be elevated in the group of inmune deficient patients as compared to normal controls. There are apparently conflicting results in the measurements of sali­ vary and sulcus fluid concentrations of antibacterial proteins. Friedman et al. (1983) found that lactoferrin in the crevicular fluid from the normal group was significantly lower than those from gingivitis group (0.63 vs. 1.5 ug/ul), but there was no differences in the lysozyme concentrations between these two groups. van Palerstein Helderman (1976) found that there were no 44 significant differences in lysozyme concentrations between normal subjects and those with gingivitis. Another important consideration in future studies of salivary anti­ bacterial proteins was that of Olson et al. (1985) who found that the sali­ va of smokers contained lower concentrations of lactoferrin and greater con­ centrations of thiocyanate than the saliva in non-smokers, and no differen­ ces in lysozyme concentrations between these two groups. It is uncertain at present whether the levels of the other antibac­ terial agents in saliva have been changed in the patients with gingivitis or chronic periodontitis. These observations may reflect differences in the methods used to measure such proteins or variabilities in the salivary secretions of such proteins in subjects with gingivitis and in smokers. The reasons for such differences remain to be explained. CHAPTER VI

SUMMARY AND CONCLUSION

SUMMARY: Ten adult clinical patients with gingivitis and ten clinically nonnal age- and sex-matched control subjects were selected for measuring the con­ centrations of lactoferrin in saliva by using an enzyme-linked inmunosorbent assay (ELISA) technique. At the baseline, all participants were given a clinical examination, and provided saliva samples. Patients with gingivitis then received the following treatment: oral hygiene instruction, scaling, root planing and gingival curettage. Approximately five weeks after the ini­ tial appointment, both patients with gingivitis and control subjects came back for reexamination. At this time, the baseline procedures were repeated, and the study tenninated.

CONCLUSION: (1) The experimental results reported in this study do show the pre­ sence of lactoferrin in the whole saliva of patients with gingivitis and clinically nonnal individuals. (2) Double antibody sandwich ELISA technique is a very sensitive and useful method to determine the concentration of lactoferrin in saliva. (3) The levels of lactoferrin in the saliva of patients with gingivi­ tis and individuals with clinically nonnal gingiva do not have significant differences as detennined by ELISA technique. This suggests_that gingivitis

45 46 does not change the host's response with respect to the lactoferrin level in saliva. (4) After the patients with gingivitis in this study received scaling, root planing, gingival curettage and oral hygiene instruction, the level of lactoferrin in their saliva did not change. This suggests that lactoferrin level in saliva does not respond to the treatment of gingivitis, even though there is a significant clinical improvement in the gingival inflanmation. BIBLIOGRAPHY

Aisen, P. and Leibman, A.: Lactoferrin and transferrin: A comparative study. Biochim. Biophys. Acta 257:314, 1972. Alfano, M.C.: The origin of gingival fluid. J. Theor. Biol. 47:127, 1974. Ambruso, D.R. and Johnston, R.B.: Lactoferrin enhances hydroxyl radical production by human neutrophils, neutrophil particulate fractions, and an enzymatic generating system. J. Clin. Invest. 67:352, 1981. Arnold, R.R., Brewer, M., and Gauthier, J.J.: Bactericidal activity of human lactoferrin: Sensitivity of a variety of microorganisms. In­ fection and Irrvnunity 28:893, 1980. Arnold, R.R., Cole, M.F., and McGhee, J.R.: A bactericidal effect for human lactoferrin. Science 197:263, 1977. Arnold, R.R., Russell, J.E., Champion, W.J., Brewer, M. and Gauthier, J.J.: Bactericidal activity of human lactoferrin: Influence of physical conditions and metabolic state of the target microorganism. Infection and Immunity 32:655, 1981. Arnold, R.R., Russell, J.E., Champion, W.J., Brewer, M. and Gauthier, J.J.: Bactericidal activity of human lactoferrin: Differentiation from the stasis of iron deprivation. Infection and Immunity 35:792, 1982. Attstrom, R.: Presence of leukocytes in crevices of healthy and chronically inflamed gingivae. J. Periodont. Res. 5:42, 1970. Attstrom, R., and Egelberg, J.: Presence of leukocytes within the gingival crevices during developing gingivitis in dogs. J. Periodont. Res. 6: 110, 1971. Baggiolini, M., DeDuve, C., Masson, P.L., and Heremans, J.F.: Association of lactoferrin with specific granules in rabbit heterophil leukocytes. J. Exp. Med. 13:559, 1970. Becraft, O.M.O., Dix, M.R., and Farmer, K.: Intramuscular iron-dextran and susceptibility of neonates to bacterial infections. In vitro studies. Arch. Oise. Child. 52:778, 1977. Bell, G.H., Davidson, J.N., Scarborough, H.: Textbook of Physiology and Biochemistry. Livingstone, London. p. 72, 1953

47 48

Benini, L., Harvey, R.F. and Vantini, I.: Lactoferrin concentration in the paroti~ saliva of patients with chronic pancreatitis. Digestion 28: 77, 1983. Biserte, G., Havez, R., and Cuvelier, R.: Les glycoprteides des secretions bronchiques. Expos. Ann. Biochim. Med. 24:85, 1963. Bishop, J.G., Schanbacher, F.L., Ferguson, L.C., and Smith, K-L.· In vitro growth inhibition of mastitis-causing colifonn bacteria by bovine apo­ lactoferrin and reversal of inhibition by citrate and high concentra­ tions of apo-lactoferrin. Infection and Inmunity 14:911, 1976.

Blanc, B., Bujard, E~, and Mauron, J.: The amino acid composition of human and bovine lactoferrin. Experientia 19:299, 1963. Blanc, B., Isliker, H.: Isolation and characterization of the red siderro­ philic protein from maternal milk : Lactoferrin. Bull. Soc. Chim. Biol. 43:929, 1961. Bornside, G.H., Bouis, P.J., and Cohn, I.: Hemoglobin and Escherichia coli, a lethal intraperitoneal combination. J. Bacterial. 95:1567, 1968. Brandtzaeg, P.: Human secretory immunoglobulins concentrations of parotid lg A and other secretory proteins in relation to the rate of flow and duration of secretory stimulus. Arch. Oral Biol. 16:1295, 1971. Buhler, E., and Muhlemann, H.R.: The gingivitis iron test. Helv. Odontol. Acta. 19:54, 1975. Bullen, J.J.: The significance of iron in infection. Rev. Infect. Dis. 3:1127, 1981. Bullen, J.J., Arnstrong, J.A.: The role of lactoferrin in the bactericidal function of polymorphonuclear leukocytes. Irrmunology 36:781, 1979. Bullen, J.J., Leigh, L.C. and Rogers, H.J.: The effect of iron compounds on the virulence of Escherichia coli for guinea pigs. Immunology 15:581, 1968. Bullen, J.J., Rogers, H.J., and Leigh, L.: Iron-binding proteins in milk and resistance to Escherichia coli infection in infants. British Medical Journal 1:69, 1972. Bullen, J.J., Rogers, H.J., and Griffiths, E.: Role of iron in bacterial infecti.on. Current Top Micro Biol Immunol. 80:1, 1978. Bullen, J.J., Wallis, S.N.: Reversal of the bactericidal effect of poly­ morphs by a ferritin-antibody complex. FEMS Letters 1:117, 1977. 49

Cantarero, L.A., Butler, J.E., and Osborne, J.W.: The adsorptive character­ istics _of proteins for polystyrene and their significance in solid­ phase inmunoassays. Anal. Biochem. 105:375, 1980. Castellino, F.J., Fish, W.W., and Mann, K.G.: Structural studies on bovine lactoferrin. J. Biological Chemistry 245:4269, 1970. Colomb, E., Pianetta, C., Estevenon, J.P., Guy, O., Figarella, C., and Sarles, H.: Lactoferrin in human pancreas. Inmunohistological local­ ization in normal and pathological pancreatic tissues. Digestion 14: 242, 1976. Dipaola, C., and Mandel, E.D.: Lactoferrin concentration in human parotid saliva as measured by an enzyme-linked inmunosorbent assay (ELISA). J. Dent. Res. 59:1463, 1980. Durr, G.H.K., Amouric, M., Bode, C., Bode, J.C., Sarles, H., Figarella, C.: Salivary secretion in chronic pancreatitis with special reference to albumin and lactoferrin. Digestion 24:87, 1982. Engvall, E., Jonsson, K., and Perlmann, P.: Enzyme-linked inmunosorbent assay II. Quantitative assay of protein antigen, inmunoglobulin G by means of enzyme-labeled antigen and antibody coated tubes. Biochim. Biophys. Acta. 251:427, 1971. Estevenon, J.P., Sarles, H., and Figarella, C.: Lactoferrin in the duodenal juice of patients with chronic calcifying pancreatitis. Stand. J. Gastroent. 10:327, 1975. Fedail, S.S., Harvey, R.F., Slamon, P.R., and Read, A.E.: Radioinmunoassay of lactoferrin in pancreatic juice as a test for pancreatic diseases. Lancet 1:181, 1978. Fields, H.A., Davis, C.L., Bradley, D.W., and Maynard, J.E.: Experimental conditions affecting the sensitivity of enzyme-linked inmunosorbent assay (ELISA) for detection of hepatitis B surface antigen (HBsAg). Bull. W.H.O. 61:135, 1983. Finkelstein, R.A., Sciortino, C.V., and McIntosh, N.A.: Role of iron in microbe-host interactions. Rev. Infect. Dis. 5:S759, 1983. Fridman, S.A., Mandel, I.D., and Herrera, M.S.: Lysozyme and lactoferrin concentration in the crevicular fluid. J. Periodontal. 54:347, 1983. Gordon, W.G., Groves, M.L., and Basch, J.J.: Bovine milk "red protein" amino acid composition and comparison with blood transferrin. Bio­ chemistry 2:817, 1963. 50

Green, I., Kirkpatrick, C.H., and Dale, D.C.: Lactoferrin specific local­ ization in the nuclei of human polymorphonuclear neutrophilic leuko­ cytes. -Proc. Soc. Exp. Biol. Med. 137:1311, 1971. Griffiths, E., Rogers, H.J., and Bullen, J.J.: Iron, plasmids and infec­ tion. Nature 284:508, 1980. Harmon, R. F. , Schanbacher, F. L. , Ferguson, L. C. , and Smith, K. L. : Concen­ tration of lactoferrin in milk of normal lactating cows and changes occurring during mastitis. Am. J. Vet. Res. 36:1001, 1975. Harmon, R.F., Schanbacher, F.L., Ferguson, L.C., and Smith, K.L.: Changes in lactoferrin, immunoglobulin G, bovine serum albumin and a-lactalbu­ min during acute experimental and natural coliform mastitis in cows. J. Infect. Immun. 13:533, 1976. Hill, G.B.: Enhancement of experimental anaerobic infections by blood, hemoglobin and hemostatic agents. Infect. Immun. 19:433, 1978. Johansson, B.: Chromatographic separation of lactalbumin from human milk whey on calcium phosphate columns. Nature 181:996, 1958. Johansson, B.: Isolation of an iron-containing red protein from human milk. Acta. Chem. Scand. 14:510, 1960. Kaki zaki, G. , Noto, N., Onuma, T. , et al.: Experimental study on the cor­ relation between the pancreas and parotid gland. Tohoku J. Exp. Med. 105:223, 1971. Kakizaki, G., Saito, T., Soeno, T., Sarahara, M., Fujimara, Y.: A new diag­ nostic test for pancreatic disorders by examination of parotid saliva. Am. J. Gastroent. 65:437, 1976. Kemeny, D.M., Urbanek, R., Samuel, D. and Richards, D.: Increased sensiti­ vity and specificity of a sandwich ELISA for measurement of lg E anti­ bodies. J. Imnunological Methods 78:217, 1985. Kirkpatrick, C.H., Green, I., Rich, R.R., and Schade, A.L.: Inhibition of growth Candida albicans by iron-saturated lactoferrin: Relation to host-defense mechanisms in chronic mucocutaneous candidiasis. J. Infections diseases 124:539, 1971. Kochan, I.: The role of iron in bacterial infections, with special consid­ eration of host-tubercle bacillus interaction. Current Top. Microbial. Immunol. 60:1, 1973. Krygier, G., Genco, R.J., Mashimo, P.A., Hausmann, E.: Experimental gingi­ vitis in Macaca speciosa Monkeys: Clinical, bacteriologic and histo­ logic similarities to human gingivitis. J. Periodontal. 44:454, 1973. 51

Konttinen, Y~T., Kulomaa, M., Malmstrom, M., Kil pi, A., and Reitamo, S. Lactoferrin in Sjogren's syndrome. Arthritis and Rheumatism 27:462, 1984. - Lankford, C.E.: Bacterial assimilation of iron. Crit. Rev. Microbiol. 2:273, 1973. Leffell, N.S., and Spitznagel, J.K.: Association of lactoferrin with lyso­ zyme in granules of human polymorphonuclear leukocytes. Infection and Irrmunity 6:761, 1972. Listgarten, M.A.: Structure of the microbial flora associated with perio­ dontal health and disease in man. A light and electron microscopic study. J. Periodontal. 47:1, 1976. Listgarten, M.A., Mayo, H.E., and Tremblay, R.: Development of dental plaque on epoxy resin crowns in man. A light and electron microsocpic study. J. Periodontol. 46:10, 1975. Loe, H.: The gingival index, the plaque index, and the retention index system. J. Periodontol. 38:610, 1967. Loe, H., and Silness, J.: Periodontal disease in pregnancy. Acta. Odontol. Scand. 21:533, 1963. Loe, H., Theilade, E., and Jensen, S.B.: Experimental gingivitis in man. J. Periodontal. 36:177, 1965. Loesche, W.J., and Syed, S.A.: Bacteriology of dental plaque in experi­ mental gingivitis. I. Relationship between gingivitis and flora. A.A.D.R. Abstract No. 108, 1975. Masson, P.L., Heremans, J.F., and Prignot, J.: Irrmunohistochemical local­ ization of the iron-binding protein lactoferrin in human bronchial glands. Experientia 21:604, 1965.

Masson, P~L., Carbonara, A.O., and Heremans, J.F.: Studies on the proteins of human saliva. Biochim. Biophys. Acta. 107:485, 1965b. Masson, P.L., Heremans, J.F., and Dive, C.H.: An iron-binding protein corrmon to many external secretions. Clin. Chim. Acta. 14:735, 1966. Masson, P.L., and Heremans, J.F.: Studies of lactoferrin, the iron-binding protein of secretions. Protides of the Biological Fluids 14:115, 1966b. Masson, P.L., Heremans, J.F., Prignot, J.J., and Wauters, G.: Inrnunohisto­ chemical localization and bacteriostatic properties of an iron-binding protein from bronchial mucus. Thorax 21:538, 1966c. 52

Masson, P.L., Heremans, J.F., Schonne, E., and Crabbe, P.A.: New data on lactoferrin, the iron-binding protein of secret;ons. Protfdes of the Biological Fluids 16:633, 1968. Masson, P.L., and Heremans, J.F.: Metal-combin;ng properties of human lac- toferrin (red milk protein). 1. The involvement of bicarbonate in the reaction. Eur. J. Biochem. 6:579, 1968b. Masson, P.L., Heremans, J.F., Schonne, E.: Lactoferrin, an ;ran-binding protein in neutrophil;c leukocytes. J. Exp. Med. 130:643, 1969. Montreuil, J., Tonnelat, J., and Mullet, S.: Preparation and properties of lactoferrin of human milk. Biochim. Biophys. Acta. 45:413, 1960. Moro, I., Umemura, S.O., Crago, S.S., and Mestecky, J.: Immunohistochemical distributfon of immunoglobulins, lactoferrin, and lysozyme in human minor salivary glands. J. Oral Path. 13:97, 1984. Mukherjee, S.: The role of crevicular fluid iron in periodontal disease. J. Periodontal. Special Issue, p. 22, 1985. Muratsu, K., and Morioka, T.: Levels of salivary lysozyme, lactoperoxidase, and lactoferrin in diabetic hamster. Infection and Immunity 48:389, · 1985. Olson, B.L., McDonald, J.L., Gleason, M.J., Stookey, G.K., Schmehorn, B.R., Drook, C.A., Beiswanger, B.B., and Christen, A.G.: Comparison of various salivary parameters in smokers before and after the use of a nicotine-containing chewing gum. J. Dent. Res. 64:826, 1985. Oram, J.D., and Reiter, B.: Inhibition of bacteria by lactoferrin and other iron-chelating agents. Biochim. Biophys. Acta. 170:351, 1968. Osease, R., Yang, H.H., Baehner, R.L., and Boxer, L.A.: Lactoferrin: A promoter of polymorphonuclear leukocytes adhesiveness. Blood 57:939, 1981. Page, R.C., and Schroeder, H.E.: Current status of the host response in chronic marginal periodontitis. J. Periodontal. 52:477, 1981. Querinjean, P., Masson, P.L., and Heremans, J.F.: Molecular weight, single­ chain structure and amino acid composition of human lactoferrin. Eur. J. Biochem. 20:420, 1971. Reitano, S., Konttinen, U.T., Segerberg, Konttinen, M.: Distribution of lactoferrin in human salivary glands. Histochemistry 66:285, 1980. 53

Reiter, B., Brock, J.H., and Steel, E.D.: Inhibition of Escherichia coli by bovine colostrum and post-colostral milk. II. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli. Inmunology 28:83, 1975. Reiter, B., and Oram, J.D.: Bacterial inhibitors in milk and other biolog­ cial fluids. Nature 216:328, 1967. Schade, A.L., and Caroline, L.: An iron-binding component in human blood plasma. Science 104:340, 1946. Schiott, C.R., and Loe, H.: The origin and variation in number of leuko­ cytes in the human saliva. J. Periodont. Res. 42:545, 1970. Skypeck, H.H., and Joseph, B.J.: Determination of trace elements in human serum by energy dispersive X-ray fluorescence. Clinical Biochem. 14: 126' 1981. Slots, J.: The microflora in the healthy gingival sulcus in man. Scand. J. Dent. Res. 85:247, 1977. Slots, J.: Subgingival microflora and periodontal disease. J. Clin. Perio­ dontics 6:351, 1979. Socransky, S.S.: Microbiology of periodontal disease. Present status and future considerations. J. Periodontal. 48:497, 1977. Socransky, S.S., Manganiello, A.O., Propas, D., Oram, V., and van Houte, J.: Bacteriological studies of developing supragingival dental plaque. J. Periodontal Res. 12:90, 1977. Sorensen, M., and Sorensen, S.P.L.: The proteins in whey. Compt. Rend. Trav. Lab. Carlsberg 23:55, 1939. Syed, S.A., Loesche, W.J., and Loe, H.: Bacteriology of dental plaque in experimental gingivitis. 2. Relationship between time, plaque score and flora. A.A.D.R. Abstract No. 109, 1975. Tabak, L., Mandel, I.D., Karlan, D., and Baurmash, H.: Alteration in lac­ toferrin in salivary gland disease. J. Dent. Res. 57:43, 1978. Tabak, L., Mandel, LD., Herrera, M. and Baumash, H.: Changes in lactofer­ rin and other proteins in a case of chronic recurrent parotitis. J. Oral Pathol 7:91, 1978b. Teuwissen, B., Masson, P.L., Osinski, P., and Heremans, J.F.: Metal combin­ ing properties of human lactoferrin: The possible involvement of Tyrosyl residues in the binding sites. Spectrophotometric titration. Eur. J. Biochem. 31:239, 1972. 54

Theilade, E., Wright, W.H., Jensen, S.B., and Loe, H.: Experimental gingi­ vitis in man. II. A longitudinal clinical and bacteriological inves­ tigation. J. Periodont. Res. 1:1, 1966. Tourville, D.R., Alder, R.H., Bienenstock, J. and Tomasi, T.B.: The human secretory irrmunoglobulin system: Invnunohistochemical localization of Ganrna A, secretory piece and lactoferrin in normal human tissues. J. Exp. Med. 129:411, 1969. Urbanek, R., Kemeny, D.M., and Samuel, D.: Use of the enzyme-linked immuno­ sorbent assay for measurement of allergen specific antibodies. J. Im­ munological Methods 79:123, 1985. van Palenstein Helderman, W.H.: Total viable count and differential count of Vibrio (campylobacter} sputorum, Fusobacterium nucleatum, Selenomonas sputigena, Bacteriodes ochraceus and Veillonella in the inflamed and non-inflamed human gingival crevice. J. Periodontal. Res. 10:294, 1975. van Palenstein Helderman, W.H.: Lysozyme concentration in the gingival crevice and at other oral sites in human subjects with and without gingivitis. Arch. Oral. Biol. 21:251, 1976. Voller, A., Bartlett, A., and Bidwell, D.E.: Enzyme inununoassays with special reference to ELISA techniques. J. Clinical Pathol. 31:507, 1978. Voller, A., Bidwell, D. and Bartlett, A.: In Mannal of Clinical Immuno­ logy. Rose, N.R., Friedman, H. p. 506, 1976. Weinberg, E.D.: Iron and susceptibility to infectious disease. Science 184:952, 1974. Weinberg, E.D.: Iron and infection. Microbial Rev. 42:45, 1978. Williams, G., and Jackson, D.S.: Two organic fixatives for acid muco­ polysaccharides. Stain Technology 31:189, 1956. Wright, D.E.: The source and rate of entry of leukocytes in the human mouth. Arch. Oral. Biol. 9:321, 1964. APPENDIX

55 56

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS - PERIODONTAL CASE RECORD

Patient I 1 Student Date Residence Address ~ Phone~ Business Address Phone - Address Phone

R

~~~~~ ~~~~~ ~~ 660~~ eo~e~ ffiG"B ~~gf~ ~~~~B ~~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 57

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS PERIODONTAL CASE RECORD Patient f 2 Student __Date _ Residence Address_,_____ Phone_ Business Address Phone _ Insurance Company Address Phone

R

twbff~~~~8 ~~t]~ij~i R 63~~60~~ eo~e~tBffi ww~~g~~ ~~~~B~~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 58

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS PERIODONTAL CASE RECORD ·- Patient I 3 Student __Date _ Residence Address-"'------Phone_ Business Address Phone _ Insurance Company Address Phone

i ffe[W(}ff ~~~~~ ~~~~ij~~~

R ~EBC(B6t}0~ eo~<:J~fBff)GB ~ JJWW~~Bf~ ~~~~B~~~ 3

PRE-EXAMINATION Date:_ POST-EXAMINATION Date: - 59

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS PERIODONTAL CASE RECORD •. Patient I 4 Student __Date _ Residence Address _____Phone_ Business Address Phone _ Phone

i JJ&&~~~B8 ~~~~~~~~

R ~ffi~~(JO<:S>~ eo~e~fBff)fiB I JJW~~~Bf~~~~~B~~\i

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 60

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS - PERIODONTAL CASE RECORD Patient I 5 Student ___ Date _ Residence Address------Phone Business Address Phone - Address Phone-

R

&&~~~~8 ~~~~~~~ R tBCCOe60~9 eo@<:}@fBE£ WtJ~~gf~ ~~~~B~~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 61

LOYOLA DENT.AL SCHOOL DEPARTMENT OF PERIODONTICS ___ PERIODONTAL CASE RECORD

Patient I 6 Student ___ Date _ Residence Address ______Phone_ Business Address ,. Phone _ Insurance Company Phone

R

{i}bfj~~3~~ ~~~~~~~ R ffi@,660~~ eo~~~fBff) WJifV~Bf~ ~~~~B~~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 62

LOYOLA DEJffAL SCHOOL DEPARTMENT OF PERIODONTICS - P E ll I 0 D 0 N T A L C A S E ll E C 0 R D

Patient I 7 Student ___ Date _ ~:!:::~\!:~~==· ------::~~=- Address Phone -

R

&~~~~~ ~~~~~~ R ~660~~ eo~e~fB ~~~g~~ ~~~~ B~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 63

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS _ PERIODONTAL CASE RECORD

Patient I 8 Student Date Residence Address --Phone-=-- Business Address_._,----,..----Phone_ Insurance Company · Address Phone

I ffe[W(ffj~~~~~ ~~~~ij~~

R ~EB~660es>~ eo9J~rJ?f9B? 1 llWW~~gf~ ~~~~B~~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 64

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS _ P E R I 0 D 0 N T A L C A S E ll E C 0 ll D

Patient f 9 Student __Date _ Residence Address ______Phone_ Business Address ' Phone _ Address Phone

R

&&~~~~~ ~~~~~~~ R ffiC(O 6 (Jo~~ e o ~ <:J '<3' fB ffi ww~~g~~ ~~~~B~~

17

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 65

- LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS PERIODONTAL CASE RECORD

Patient f 10 . c Student ___ Date __ Residence Address ______Phone_ Business Address Phone _ Address Phone

PRE-EXAMINATION Date: --- POST-EXAMINATION Date: -- 66

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS - PERIODONTAL CASE RECORD

Patient I 11 Student ---Date_ :::~::::e~::~::• _..______:~~:: - Address Phone-

R

&&~~~~~ ~~~~~~~ R ffie()j 6 6 0 C5)~ e 0 ~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 67

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS - P E ll I 0 D 0 N T A L C A S E ll E C 0 ll D Patient I 12 Student ___ Date llesidence Address --'--"'------Phone-- Business Address Phone -- Insurance Company Address Phone

PRE-EXAMINATION Date: --- POST-EXAMINATION Date: --- 68

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS ___ PERIODONTAL CASE RECORD

Patient I 13 Student __Date _ Residence Address ______Phone_ Busi'ness Address ' Phone _ Insurance Company Address Phone

I ffe[Wbfj~~~~~ ~~~~~~~

R ~ ffie(B660~~ eof;;e~efBre I J?WW~~gf~ ~~~~B~~

PRE-EXAMINATION Date: __ POST-EXAMINATION Date: -- 69

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS - PERIODONTAL CASE RECORD Patient I 14 Student ___ Date _ Resi~ence Address~,------Phone_ Business Address Phone _ Address Phone

R

17 PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 70

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS - PERIODONTAL CASE RECORD Patient I 15 Student ___ Date Residence Address------Phone­ Buiiness Address Phone - Address Phone

R

tw(;ff~~~~8 ~~~~~~~ R ffi~6(J0~~ eo~<:Jrc?ef9f£ ww~~Bf~ ~~~~B lfi~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 71

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS PERIODONTAL-CASE RECORD

Patient # 16 Student __Date_ Residence Address------Phone_ Business Address Phone Address Phone-

R

[i}bfj~~~~8 ~~~~~~~ R ffi~660~ eo~<:J~fBffi ww~~Bf~ ~~~~B~~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: --- 72

I.OYOLA DENTAL SCHOOL DEPARTMENT OF PERIOOONTICS - PERIODONTAL CASE RECORD Patient f 17 Student ___ Date _ ReJidence Address ______Phone_ Business Address Phone _ Inaurance Company Address Phone

R

Jl

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- 73

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS - PERIODORTAL CASE RECORD

Patient I 18 Student ___ Date __ llesi@nce Address------Phone __ Business Address Phone __ Insurance Company Address Phone

R

R

PRE-EXAMINATION Date: --- POST-EXAMINATION Date: --- 74

LOYOLA DENTAL SCBOOL DEPARTMENT OF PERIODONTICS PERIODONTAL CASE RECORD

Patient I 19 Student ___ Date_ Residence Address------Phone_ Business Address Phone _ Insurance Company Address Phone

R

&&~~~~~ ~~~~~~~ R EB~6(10~~ eo~<::J'

PRE-EXAMINATION Dat:e: -- POST-EXAMINATION Date: -- 75

LOYOLA DENTAL SCHOOL DEPARTMENT OF PERIODONTICS PERIODONTAL CASE RECORD

Patient f 20 Student Date Residence Address " --Phone- Business Address ------Phone - Address Phone

&bff~~~~8 ~~~~ij~% R ffi~66a0es>9 eo~e@tBf£ W~~~gf~ ~~~~B~~

PRE-EXAMINATION Date: -- POST-EXAMINATION Date: -- APPROVAL SHEET

The thesis submitted by Hwu-Rang Lin has been read and approved by the following committee:

Dr. Patrick·Toto, Director Professor, Gen/Oral Pathology, Loyola Dr. Anthony Gargiulo Professor, Periodontics, Loyola Dr. James Hagen Associate Professor, Microbiology, Loyola

The final copies have been examined by the director of the thesis and the signature which appears below verifies the fact that any necessary changes have been incorporated and that the thesis is now given final approval by the Committee with reference to content and form. The thesis is therefore accepted in partial fulfill­ ment of the requirements for the degree of Master of Science.

//. .. .~£)~ Date ~.~~ature

76